1. Field of the Invention
The invention relates to β-galactosidase fragment as a label.
2. Background Information
β-galactosidase has found wide use as a label in a variety of environments. Both the intact enzyme and fragments thereof have been used as a label in the determination of the presence of an analyte, interactions between different molecules, and the like. The enzyme is very versatile in having a high turnover rate, fluorescent, luminescent and light absorbent products from its catalyzed reactions, stable and active under a variety of conditions.
Because of its versatility, new applications are of great interest. In one application, one is interested in using the small fragment as a label fused to another protein. Such fusion products can find application in many situations, particularly intracellular situations, where one is interested in the fused protein accurately mimicking the activity of the natural protein. Desirably, the β-galactosidase fragment should be small, so as to provide the minimal interference with the activity, transport and interactions with other proteins. Heretofore, the small fragment has been greater than 40 amino acids, 43 amino acids having been identified as being active. Based on this disclosure it was not certain that one could further truncate the small fragment and obtain a complex with the large fragment that would have a sufficient turnover rate so as to be useful as a label.
The proteomics field is rapidly moving toward determining of the function of proteins, including their interaction, degradation and modulation. Consequently, a technology capable of measuring the function at low expression levels, particularly those levels at which proteins are expressed endogenously, is required for extensive deployment of functional proteomics in drug discovery. Enzyme fragment complementation (EFC) technology provides one such platform for addressing not only expression of proteins but also for deciphering protein-protein interactions. EFC is a generic term to describe the combination of enzyme fragments to form active enzyme, followed by detection of that activity by measurement of an hydrolysis product, generally by colorimetric, fluorometric or chemiluminescent methods. It has the advantage of providing an amplification step, due to enzyme turnover, as part of the detection system.
In one aspect of EFC, the fragments of the enzyme have sufficient affinity for each other to complex to form an active enzyme without relying on the affinity of the binding of complementary pairs to which the fragments are attached. This capability has been amply demonstrated with β-galactosidase, using a small enzyme donor (ED) fragment and a large enzyme acceptor (EA) fragment
The enzyme donors that have been typically used in CEDIA® or EFC are typically 90 mers (amino acids), for example ED4, ED14 and ED28 are 90 mers with one cysteine, one cysteine plus one lysine and two cysteines respectively. These amino acids serve as handles for conjugating various molecules to the enzyme donor. The enzyme donors which are currently used in marketed CEDIA products are made by fermentation of genetically engineered bacteria, which need several processing steps culminating in a tedious purification of the product ED. This approach has worked for production purposes till now.
Advances made in peptide synthesis chemistry does enable 90 mers to be synthesized in useful amounts, however, the cost is still high when compared to the site directed mutagenisis approach of making ED variants. Shorter EDs (fewer than 50 mers) would make the synthetic EDs commercially competitive. Automated peptide synthesis is now routine in many laboratories, typically a 40-50 mer can be synthesized in a week. A disadvantage with the genetic approach to produce ED variants is that it is extremely time consuming and labor intensive and it takes the same amount of time to produce a 50 mer as a 90 mer which is in the vicinity of 6-8 weeks. From this perspective shorter synthetic EDs are attractive, first for research uses as variants can be synthesized with a turnaround time of a week (for 40 mers) and second the cost for scale up makes it competitive with the recombinant approaches for commercial use. The relative ease and flexibility with which shorter ED variants can be made by synthetic approaches makes it possible to study structure activity relationships to determine sequences that are essential for complementation activity. Since ED conjugates tend to alter complementation depending on the nature of the ligand that is appended, such structure activity relationships can be exploited to yield conjugates with improved performance.